Regulation of the Sag Operon
The sag Operon The sag operon is a genetic sequence in Group A Streptococci (GAS) that codes for a very harmful virulence factor called Streptolysin S (SLS). Streptolysin S is the responsible agent for beta-hemolysis seen on blood agar cultures in a clinical setting. This protein has cytolytic activity which disrupts the membranes of cells by forming pores, and is formed by a chain of 53 amino acids. The sag locus, containing sagA through sagI (9 total), is required for effective SLS production. It has been proved that genetically manipulating any part of the sag locus will completely inactivate the hemolytic activity. The sagA gene is the most important gene in SLS production because it codes for the structural protein of the SLS molecule. The sagB,C, D gene codes for modifying enzymes while sagG,H,I codes for transport proteins for sagA,B,C. Mechanisms Control of SLS does not occur on a regular basis, however, the sag operon can be repressed by Catabolite Control Protein A (CcpA). CcpA is a mediator of Carbon Catabolite Repression (CCR), and in GAS strains it has the greatest effect on the sag operon. CCR is used by GAS strains to target the enzymes and transport proteins needed for the obtaining alternative carbon sources when needed. CCR is primarily used for controlling the bacterial cells ability to uptake different kinds of sugars needed for energy, but on occasion it has a tremendous effect on virulence regulation. In the case of SLS production, the CcpA is able to recognize the catabolite response element (cre) that is found within the sagA promoter sequence. Along with the cre found in the sag locus, there is also a pel regulatory RNA. '''Pel stands for "pleiotropic effect locus," which contains the sagA gene. The sagA/pel sequence is transcribed in order to produce SLS, but is repressed by CcpA mediated CCR. The CcpA is known to directly bind to the sagA/pel region of the gene by forming a complex with a metabolic sensor protein in a phosphoenolpyruvate phosphotranferase system. This protein called HPr, is phosphorylated on a serine residue which allows it to create the '''HPr-CcpA complex. After this occurs, the complex can now bind to the cre within the promoter region of the gene. Because of this, SagA/pel is repressed during growth phases of the bacterial cell. The operon can also be repressed by a two component system in GAS called CovRS. The S component of this system is a sensor kinase that phosphorylates CovR depending on environmental conditions. CovR is the response regulator that negatively regulates virulence genes. This negative regulation means that the gene expression for SLS is repressed. Molecular and Environmental Stimuli Expression of the sag operon is under molecular and environmental stimuli. One of the greatest effects on a bacterial cell's activity is nutrient availability. The role of the cytolytic ability of GAS is to obtain nutrients from the surrounding environment, specifically from the host cell. The main function of CcpA is to down-regulate when glucose in the environment is abundant. When there are high quantities of fuel source (like glucose), the repression of the sag locus is preferred.When there are low quantities of fuel sources, the sag locus is transcribed because the bacterial cell must sustain its energy needs. If the gene is not being repressed, the cytolytic activity of SLS will cause host-cell damage and induce the recruitment of white blood cells. This is not desirable because when there is plenty of fuel available for uptake, the bacterial cell has no need for cytolytic activity. This idea proves that the regulation of the sag operon is monitored closely, and must be monitored closely to cause virulence when it is needed. Biological Reason for Gene Expression The sag operon is repressed during growth phases and does not become active until the cell reaches a stationary phase. This is a key point because as the cell reaches the stationary phase, it must begin to obtain energy for further replication. Thus, the expression of the sag operon is essential during late stages of the growth phase and early stages of the stationary phase to cause red blood cell hemolysis and nutrient acquisition. Glucose and other sugars for a carbon source aside, Iron is a biological necessity for cells. When the red blood cells are lysed, iron becomes available for uptake via other mechanisms that are under different controls. If the sag operon is transcribed to create SLS, GAS can use this iron for its own good for cell growth. References 1. Lecture #2: Streptococci by Dr. Doug Johnson, MMG 222, Spring Semester 2014, University of Vermont 2. CcpA-Mediated Repression of Streptolysin S Expression and Virulence in the Group A Streptococcus PMCID: PMC2493232 3. Genetic Locus for Streptolysin S Production by Group A Streptococcus PMCID: PMC101736 4. Virulence control in group A Streptococcus by a two-component gene regulatory system: Global expression profiling and in vivo infection modeling (Proceedings of the National Academy of Sciences of the USA)